Method of covalent coupling

ABSTRACT

A method of preparing conjugates by means of covalent coupling of a polysaccharide selected from heparin, heparin derivatives, at least partially deacerylated dermatan sulphate and dextran sulphate and a solid substance containing primary amino groups, said method comprising the following steps: a) reduction of the saccharide so that its terminal monosaccharide unit is converted to an alditol; b) periodate oxidation of the alditol formed in step a) to the formation of a terminal aldehyde group under cleavage of the monosaccharide unit between two vicinal groups selected from hydroxyl and amino groups; and c) reductive coupling of the alditol via the aldehyde group to the amino group of the solid substrate.

This is the National Phase Application of PCT/SE00/02198 filed Nov. 9,2000.

TECHNICAL AREA

The present invention relates to the manufacture of conjugates betweenoligo- or polysaccharides and aminated substrates. The invention alsocovers the products hereby obtained.

BACKGROUND OF THE INVENTION

Covalent coupling of saccharides to substrates can be made mainly in twodifferent ways, on the one hand through so-called “end-point attachment”(EPA), on the other hand by so-called “multipoint attachment” (MPA). Inthe first case the saccharide is coupled via its terminal reducingmonosaccharide unit. In the second case, which is relevant withpolysaccharides, it is coupled via several monosaccharide units insidethe polysaccharide. Most of the high molecular carbohydrates present innature, which are coupled to some other high molecular compound, areimmobilized via their reducing monosaccharide unit, and such naturalconjugates are for example glycoproteins, glycolipides, proteoglycinesand lipopolysaccharides.

Coupling via “end-point attachment” means contrary to “multipointattachment” that the molecule in an immobilized state to a high degreemaintains its natural conformation and thereby maintains its capacity tospecifically interact with other molecules, such as plasma proteins,growth factors, antibodies, lectins and enzymes.

For reductive coupling to aminated substrates of polysaccharides one hastried to make use of the aldehyde functions in terminal reducingmonosaccharide units. However, these aldehyde functions are present ashemi-acetals, which means that the aldehyde function has extremely lowreactivity resulting in unacceptably low coupling yields in most cases.

A useful method for “end-point attachment” of polysaccharides toaminated surfaces is described in U.S. Pat. No. 4,613,665.

This known method is briefly constituted by partial degradation of apolysaccharide-containing D-glucosamine or D-galactosamine units, forexample heparin, with sodium nitrite in an acid environment. Thisresults in the formation of 2,5-anhydro-D-mannose units as reducingterminal units. This reducing terminal unit carries a reactive aldehydefunction, which is not engaged in any hemi-acetal formation and,accordingly the aldehyde group can in high yield by reductive aminationbe coupled to substrates containing primary amino groups.

This known method is associated with certain drawbacks, among which thefollowing can be mentioned.

The method requires that the polysaccharide contains D-glucosamine orD-galactosamine units with non-derivatizing and thereby primary aminogroups capable of diazotization.

In the activation the molecule is degraded which for certainapplications results in lost or substantially reduced biologicalactivity.

The method also means that only half of the molecules formed in thedegradation obtain a reducing end capable of coupling.

SUMMARY OF THE INVENTION

The present invention has for a main object to provide a new methodwhereby the disadvantages of the prior art are eliminated or at leastessentially reduced.

Yet another object of the invention is to provide a method in which thesubstance subject to coupling to an aminated substrate will not bedegraded or damaged in connection to the coupling procedure, whereby theproperties of the substance are maintained substantially unchanged afterthe coupling to the substrate has taken place.

Yet another object of the invention is to provide a method where allmolecules are made subject to coupling, whereby the yield in thecoupling reaction is substantially improved compared to the prior art.

A further object of the invention is to provide a process which involves“end-point” coupling to aminated substrates.

For these and other objects which will be clear from the followingdisclosure there is provided through the present invention a method ofpreparing conjugates by means of covalent coupling of a polysaccharideselected from heparin, heparin derivatives, at least partiallydeacetylated dermatan sulphate and dextran sulphate and a solidsubstance containing primary amino groups. Accordingly, the methodaccording to the invention is a method of heterogeneous charactercharacterized by the following steps:

a) reduction of the saccharide so that its terminal monosaccharide unitis converted to an alditol;

b) periodate oxidation of the alditol formed in step a) to the formationof a terminal aldehyde group under cleavage of the monosaccharide unitbetween two vicinal groups selected from hydroxyl and amino groups; and

c) reductive coupling of said alditol via the aldehyde group to theamino group of the solid substrate.

The reduction in step a) above is suitably carried out with the reducingagent selected from borohydrides, catalytic reducing agents and Raneynickel. It is particularly preferred to carry out the reduction in stepa) with a borohydride, especially sodium borohydride or potassiumborohydride.

With regard to the oxidation in step b) above it is suitably carried outwith periodic acid or a sodium or potassium salt thereof.

The reductive coupling in step c) above is suitably carried out with acyanoborohydride, for example sodium cyanoborohydride or potassiumcyanoborohydride.

A preferred embodiment of the method according to the invention thusmeans that the reduction in step a) is carried out with sodiumborohydride, the oxidation in step b) is carried out with sodiumperiodate and the reductive coupling in step c) is carried out withsodium cyanoborohydride.

The method according to the present invention is particularly suited tobe used in connection with polysaccharides possessing biologicalactivity. Thus, the polysaccharide may be selected from heparin, heparinderivatives, at least partially deacetylated dermatan sulphate anddextran sulphate.

Particularly preferred polysaccharides are heparin and heparan sulphate.

With regard to the character of the substrate there may be mentioned asexamples of substrates aminated plastic objects, for example havingsurface coatings of polyethylenimine or chitosan. Preferred surfacecoating is chitosan which in its capacity as a deacetylated chitincontains primary amino groups suited for the coupling techniquesinvolved in the present invention.

The invention also includes conjugates made by the method defined above.

The reactions involved in the method according to the present inventionwill now be further elucidated for a better understanding of thereaction mechanisms.

The periodate oxidation in step b) above involves the formation of acyclic ester which is then cleaved and inter alia results in twoaldehyde functions according to formula I below.

Cleavage of vicinal dioles by periodate oxidation is previously knownbut as far as is known it has not previously been applied to saccharideimmobilization in accordance with the present invention. In the reactionsteric factors have a decisive influence on the rate by which differentvicinal dioles are cleaved by periodate.

Thus, vicinal OH-groups in furanosides and pyranosides which havecis-orientation are more rapidly cleaved than such havingtrans-orientation. Certain trans-dioles in rigid systems are evenresistant to periodate oxidation. Acyclic dioles are generally oxidizedmore rapidly than cyclic compounds, and such of threo-configuration aremore rapidly oxidized than such with erythro-configuration.

The invention will even if it is not restricted hereto be exemplified inthe following with reference to heparin. Formula II below describes arepresentative part of the interior of the heparin molecule. The leftD-glucosamine unit wherein R is acetyl, cannot be oxidized withperiodate. Of the other units only the glucuronacid unit is capable ofoxidation but contains, however, dioles oriented trans-di-equatoriallyand difficult to oxidate.

The major part of the molecules in native heparin having a molecularweight of between about 7000 and about 25000 contains terminally thefollowing sequence:

- - - 4) -α-D-GlcpNR- (1 - - - 4) -β-D-GlcA,

wherein R is acetyl or —SO₃H.

The terminal group can be reduced to the alditol of formula III below,the pH value suitably being maintained so high as not to lactonize theacid. By cautious periodate oxidation the threo-glycol grouping ispreferably oxidized and a compound of formula IV containing a terminalaldehyde group will be obtained.

The reaction can possibly proceed further to the formation of analdehyde function of C-atom 5.

The relevant polysaccharides are then coupled to a solid substratecontaining primary amino groups by reductive amination in accordancewith formula V below.

In an analogous manner for example chitosan can be immobilized by“end-point” -coupling according to formula VI below.

Here vicinally oriented amino and hydroxyl groups are oxidized byperiodate according to formula VII below.

The reactions involved in the present invention are by formulae shown onthe appended scheme. P within a ring designates the polymer or thepolysaccharide, whereas only the structure of the terminal unit has beenillustrated. COOH in 6-position refers to polysaccharides having aterminal glucuronic acid unit, whereas CH₂OH in 6-position relates topolysaccharides having glucosamine units in terminal position.

The coupling techniques according to the present invention constitute anessential contribution to the possibility of immobilizingpolysaccharides to aminated solid substrates while maintaining theoriginal properties of the saccharide, for example biologic activity.

The invention will in the following be illustrated further bynon-limiting examples, wherein parts and percentages relate to weight ifnot otherwise stated.

EXAMPLE 1 Reductive-oxidative Activation of Native Heparin

Sodium borohydride (225 mg) is dissolved in distilled water (150 mL).The sodium salt of native heparin (Pig mucosa, Kabi Vitrum, 3 g) isadded, and the reaction mixture is allowed to stand under stirring for18 hours. After dialysis against 10 L distilled water for 18 hours thereduced heparin is freeze-dried and the yield is 2,9 g.

The reduced heparin (2,9 g) is dissolved in 250 mL imidazole buffer, 40mM, pH 6,5. Sodium periodate (640 mg) is also dissolved in 250 mLimidazole buffer 40 mM, pH 6,5. The two solutions are mixed and areallowed to stand in a flask enclosed in foil at 0° C. on ice understirring for 5 minutes. Then 1 mL glycerol is added for the purpose ofdestroying the excess periodate, and the solution is allowed to standunder stirring for 1 hour. The reaction mixture is then dialysed against10 L distilled water for 18 hours and the heparin is finallyfreeze-dried. The yield of activated heparin (RO-heparin) is 2,4 g.

EXAMPLE 2 Decomposition of Heparin With Nitrous Acid

Heparin (Pig mucosa, Kabi Vitrum) is dissolved in water (300 ML). Thesolution is cooled to and maintained 25 at 0° C. in ice-water. Thenfirst sodium nitrate (NaNO₂, 10 mg) and then acetic acid (2 mL) areadded to the solution under stirring. The reaction mixture is maintainedat 0° C. for 2 hours, dialysed and freeze-dried. The yield of degradedheparin is 0,7 g.

EXAMPLE 3 Periodate Oxidation of Native Heparin

In a typical example, where 10% of the monosaccharide units in heparinare to be oxidized, native heparin (Pig mucosa, Kabi Vitrum, 5 g) isdissolved in water (100 mL) and sodium periodate (0,5 g) is added. Thesolution is kept in the dark at room temperature for 24 hours. Thereaction mixture is dialysed against distilled water and freeze-dried tothe formation of 4,2 g heparin derivative containingdialdehyd-functions.

EXAMPLE 4 Amination of a Polyethylene Surface With Polyethylenimine(PEI)

A polyethylene surface (polyethylene film) is treated for 2 minutes atroom temperature with 2% (w/v) solution of sodium permanganate inconcentrated sulphuric acid and is then carefully rinsed with distilledwater. The polymer surface is treated at room temperature withpolyethylenimine SN (PEI, 0,05% in borate buffer, pH 9,0) together withcrotonaldehyde (0,034% in borate buffer, pH 9,0).

The polymer surface is carefully rinsed with large volumes of distilledwater and treated with the solution of dextran sulphate (Pharmacia) 0,1g/L in 0,15 M NaCl, pH 3,0, for 10 minutes at 55° C. The polymer surfaceis again carefully rinsed with large volumes of distilled water andtreated once again at room temperature with polyethylenimine SN (PEI,0,05% in borate buffer, pH 9,0) together with crotonaldehyde (0,034% inborate buffer, pH 9,0). The treatment with dextran sulphate according tothe above is then repeated and the surface is carefully rinsed withdistilled water and treated with a solution of 0,05% PEI at pH 9,0(adjusted with 1 M NaOH) for 10 minutes at room temperature and thenagain washed. The presence of amino groups is verified with an indicator(poinceau S, Sigma).

EXAMPLE 5 Amination of Polyethylene Surface With Chitosan

A polyethylene surface is treated in the same manner as in Example 4with the difference that a solution of chitosan having a degree ofN-acetylation of 15% (0,25 w/v) in water is used instead of PEI and thatthe surface after treatment with this solution is rinsed with ethanol(80% v/v) instead of water. The presence of amino groups is verified inthe same manner as in Example 4.

EXAMPLE 6 Coupling of RO-heparin to Flexible Polyethylene Tubings

Polyethylene tubings (1 m, 1,8 mm inner diameter) are aminated in themanner described in Example 4. The tubings are then treated for 2 hoursat 50° C. with RO-heparin prepared in accordance with Example 1 (500 mL,0,025% w/v) and sodium cyanoborohydride (0,003% w/v in 0,15 M NaCl, pH3,9) and are washed with distilled water and finally with borate buffer,pH 9,0. The presence of heparin on the polyethylene tubings isvisualized using an indicator, Toluidine blue, giving a lilac colour inthe presence of sulphated polysaccharides.

EXAMPLE 7 Coupling of Nitrite-degraded Heparin to Flexible PolyethyleneTubings

This coupling is carried out in accordance with Example 6 above with themodification that nitrite-degraded heparin prepared in accordance withExample 2 is used in the coupling. With regards to details concerningthis coupling technique reference is made to U.S. Pat. No. 4,613,665.

EXAMPLE 8 Coupling of Periodate Oxidized Heparin to FlexiblePolyethylene Tubings

This coupling is carried out in accordance with Example 6 above but withthe modification that periodate-oxidized heparin prepared in accordancewith Example 3 is used in the coupling procedure.

EXAMPLE 9 Comparing Studies of AT-uptake

The ability of the heparin surface to bind antithrombin (AT) reflectsthe density of heparin molecules having unaffected AT-binding sequencesand constitutes a measure of the non-thrombogenic character of thesurface. The larger the ability to bind antithrombin the better thesurface is with regard to the biological activity of the heparin.

TABLE Control* Ex 6 Ex 7 Ex 8 AT 0, 02 2, 8 2, 6 0, 9 pmol/cm² *controlusing untreated tubing

EXAMPLE 10 Coupling of Reductively-oxidatively Activated DextranSulphate to Polyethylene Film

Sodium borohydride (225 mg) is dissolved in distilled water (150 mL).The sodium salt of dextran sulphate (4 g, Pharmacia) is added and thereaction mixture is allowed to stand under stirring for 18 hours. Afterdialysis against 10 L distilled water for 18 hours the reduced dextransulphate (heparinoide) is freeze-dried and the yield is 3,5 g. Thereduced dextran sulphate (3,0 g) is dissolved in 250 mL imidazolebuffer, 40 mM, pH 6,5. Sodium periodate (640 mg) is also dissolved in250 mL imidazole buffer, 40 mM, pH 6,5. The two solutions are mixed andallowed to stand in a flask wrapped in foil at 0° C. on ice understirring for 5 minutes. Then 1 mL glycerol is added for the purpose ofdestroying the excess periodate, and the solution is allowed to standunder stirring for 1 hour

The reaction mixture is dialysed against 10 L distilled water for 18hours, and the product is finally freeze-dried. The yield ofRO-heparinoide is 2,5 g.

The RO-heparinoide is coupled to an aminated polyethylene film preparedin accordance with Example 4, the coupling is carried out in the samemanner as described in Example 6. The presence of dextran sulphate onthe polyethylene film is visualized using an indicator, Toluidine blue,giving a lilac colour in the presence of sulphated polysaccharide. Thecoupling yield is about 5 mg/cm² (determined using FTIR).

The invention is not restricted to the above embodiments and examplessince the method is applicable to covalent coupling of all types ofoligo- and polysaccharides having a terminal reducing monosaccharideunit to all forms of substrates containing primary amino groups.

What is claimed is:
 1. A method of preparing conjugates by means ofcovalent coupling of a polysaccharide selected from heparin, heparinderivatives, at least partially deacetylated dermatan sulphate anddextran sulphate and a solid substance containing primary amino groups,comprising the steps: a) reduction of the saccharide so that itsterminal monosaccharide unit is converted to an alditol; b) periodateoxidation of the alditol formed in step a) to the formation of aterminal aldehyde group under cleavage of the monosaccharide unitbetween two vicinal groups selected from hydroxyl and amino groups; andc) reductive coupling of said alditol via the aldehyde group to theamino group of the solid substrate.
 2. A method according to claim 1,characterized in that the reduction in step a) is carried out with areducing agent selected from borohydrides, catalytic reducing agents,and Raney nickel.
 3. A method according to claim 2, wherein thereduction in step 2) is carried out with a borohydride.
 4. A methodaccording to claim 1, wherein the oxidation in step b) is carried outwith periodic acid or a sodium or potassium salt thereof.
 5. A processaccording to claim 1, wherein the reductive coupling in step c) iscarried out with a cyanoborohydride.
 6. A method according to claim 1,wherein the reduction in step a) is carried out with sodium borohydride;the oxidation in step b) is carried out with sodium periodate; and thereductive coupling in step c) is carried out with sodiumcyanoborohydride.
 7. A method according to claim 1, wherein thesaccharide is a polysaccharide which is biologically active.
 8. A methodaccording to claim 7, wherein the polysaccharide is heparin or heparansulphate.
 9. A method according to claim 1, wherein the substrate isselected from polymeric objects aminated on the surface thereof.
 10. Amethod according to claim 1, wherein the substrate is provided with acoating of polyethylenimine or chitosan.
 11. Conjugate made by themethod according to claim
 1. 12. A method according to claim 2, whereinthe oxidation in step b) is carried out with periodic acid or a sodiumor potassium salt thereof.
 13. A method according to claim 3, whereinthe oxidation in step b) is carried out with periodic acid or a sodiumor potassium salt thereof.
 14. A process according to claim 2, whereinthe reductive coupling in step c) is carried out with acyanoborohydride.
 15. A process according to claim 3, wherein thereductive coupling in step c) is carried out with a cyanoborohydride.16. A process according to claim 4, wherein the reductive coupling instep c) is carried out with a cyanoborohydride.
 17. A method accordingto claim 2, wherein the saccharide is a polysaccharide which isbiologically active.
 18. A method according to claim 2, wherein thesubstrate is selected from polymeric objects aminated on the surfacethereof.
 19. A method according to claim 2, wherein the substrate isprovided with a coating of polyethylenimine or chitosan.
 20. Conjugatemade by the method according to claim
 2. 21. A method according to claim3, wherein the borohydride is sodium borohydride or potassiumborohydride.